The present invention relates to nucleotide sequences encoding the tomato light hypersensitive phenotype, encoded proteins and uses thereof.
In particular the present invention relates to nucleotide sequences encoding a protein, whose qualitative or quantitative modification and/or inhibition in plants induces high levels of carotenoids and/or flavonoids and/or chlorophylls, in comparison with wild-type plants; the invention also relates to the use of these nucleotide sequences for the production of engineered plants to be employed in the agro-industrial sector.
Light is a critical environmental signal that controls many aspects of plant growth and development. It is perceived by a sophisticated series of photoreceptors: the phytochromes, which absorb red and far red light, the cryptochromes, which absorb blue and UV-A light wavelengths, and the UV-B receptors (Mustilli and Bowler, 1997). Together with endogenous hormonal signals, these photoreceptors regulate the developmental changes known as photomorphogenesis. Photomorphogenesis is defined as the influence of light on plant development and comprises leaf and chloroplast development and the regulation of photosynthetic apparatus components, by means of the coordinated expression of both nuclear and cytoplasmic genes. Moreover, due to the light response, photoprotectant pigments such as flavonoids are also produced. The modifications occurring during photomorphogenesis have been characterized by studying light effects on Arabidopsis seedling development (von Arnim and Deng, 1996). Light-grown Arabidopsis seedlings display short hypocotyls, open and expanded cotyledons and the expression of light-regulated genes which are responsible for flavonoid and chlorophyll biosynthesis (e.g. chalcone synthase, CHS; chlorophyll A/B binding protein, CAB). Dark-grown seedlings display elongated hypocotyls, closed cotyledons and repression of light-regulated genes.
In higher plants, the phytochromes are encoded by a gene family (e.g. PHYA-E in Arabidopsis, Sharrock and Quail, 1989; Clack et al., 1994) and although they are the best characterized photoreceptors, relatively little is known about how the light signals perceived by phytochromes are transduced to the nucleus to activate the various developmental, physiological and molecular responses to light. Recently, biochemical studies using microinjection into cells of the phytochrome deficient aurea (au) tomato mutant, along with pharmacological studies in photomixotrophic soybean cell cultures, have implicated heterotrimeric G-proteins, cGMP, calcium and calmodulin as intermediates in phytochrome signal transduction pathways (Bowler and Chua, 1994; Mustilli and Bowler, 1997). In parallel, several genetic screens have been developed to identify mutants potentially affected in light signal transduction (Chamowitz and Deng, 1996). Most of the photomorphogenic mutants have been characterised in Arabidopsis and can be classified as either insensitive or constitutive mutants. Insensitive mutants display a light-blind elongated phenotype in the light. Some are mutated in the photoreceptors themselves, whilst others are presumed to encode positive regulators of light signal transduction pathways (Chamovitz and Deng, 1996; Chory et al., 1996; Whitelam and Harberd, 1994). Conversely, constitutive de-etiolated mutants (e.g. cop/det/fus/cpd) display light grown morphologies when grown in the dark together, in some cases, with the inappropriate expression of light regulated genes such as CAB and CHS (Millar et al., 1994; Szekeres et al., 1996). The recessive nature of these mutations suggests that they are loss-of-function and that the wild-type genes are repressors of photomorphogenesis in darkness. However, although epistasis tests with phytochrome-deficient mutants have indicated that they function downstream of phytochrome, they are not specifically mutated in phytochrome signal transduction because many have altered tissue specificities as well as other additional phenotypes not directly related to light (Mayer et al., 1996; Chory and Peto, 1990; Millar et al., 1994; Szekeres et al., 1996). It is therefore not clear how COP/DET/FUS/CPD proteins function in the signal transduction pathways defined biochemically (Bowler and Chua, 1994).
A more targeted approach to identify specific components of signal transduction pathways specific for phytochrome could be the isolation of mutants with altered dynamics of light responses, rather than mutants with constitutive phenotypes in the absence of light. Several such light hypersensitve mutants have already been isolated in tomato (denoted hp-1, hp-2, atv, Ip; Kendrick et al., 1994). In particular, the recessive non-allelic hp-1 and hp-2 mutants have been characterized by their exaggerated light responsiveness, displaying higher anthocyanin levels (a flavonoid subgroup), shorter hypocotyls and more deeply pigmented fruits than wild-type plants. These mutants were first identified in 1917 (Reynard, 1956) and in 1975 (Soressi, 1975), respectively. Recently, hp-1w (Peters et al., 1989) and hp-2j (Van Tuinen et al., 1997) mutants have been isolated and identified as new hp-1 and hp-2 alleles, respectively. Because these phenotypes appear to be identical to those obtained by ectopic expression of phytochrome A (PHYA) in tomato (Boylan and Quail, 1989), it would appear that the hp mutation may affect fairly specifically phytochrome responses. The recessive nature of the mutations, coupled with results from epistasis tests of hp-1 with the phytochrome deficient tomato mutants aurea (au), phyA (fri), and phyB (tri), have suggested that HP genes encode negative regulators of light signal transduction mechanisms, acting downstream of both PhyA and PhyB (Kerckhoffs et al., 1997). The fact that no counterparts of hp mutants have been isolated so far in Arabidopsis, along with the observation that in tomato anthocyanin production and the expression of photoregulated genes (e.g., CHS and CAB) is strictly light-dependent, indicates the importance of hp mutants for studying phytochrome-dependent signal transduction. Furthermore, microinjection-based studies using the au tomato mutant have shown that tomato is an excellent model system to map the role of individual components in the phytochrome activated signalling cascade (Bowler and Chua, 1994). Therefore the identification and characterization of hp genes is likely to be very important for studying the regulation of photomorphogenesis in plants.
The authors of the present invention have cloned the tomato HP-2 gene and have studied at the molecular level the role of the HP-2 protein during the modulation of photomorphogenesis and fruit development. The authors have found that the tomato HP-2 gene exhibits high sequence homology with the Arabidopsis DET1 gene, which belongs to the above described constitutive COP/DET/FUS mutant group. Therefore the tomato HP-2 gene has been renamed TDET1.
The authors have used Solanum lycopersicum (tomato) species plants, but those skilled in the art will recognize that the cloning could be repeated with no inventive efforts with other plant species, as but not limited to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species. The authors have cloned and sequenced the gene responsible for the tomato hp mutation (high pigment), which causes a light hypersensitive phenotype, thus enhancing carotenoid, and/or chlorophyll and/or flavonoid pigment levels. The gene is the first to be identified that causes such a mutant phenotype.
hp mutants potentially have a direct application in the agro-industrial sector, for generating tomato fruits with high carotenoid and/or flavonoid contents. In particular, in tomato fruits of hp mutants, a high content of the carotenoid lycopene as well as other carotenoids and flavonoids has been observed(Thompson, 1955; Yen et al., 1997). However, up to now the use of hp mutants in the agro-industrial sector, even if bred into various commercial varieties, has been impaired because of the fact that the hp mutation generates other undesirable phenotypes, such as reduced internode length and reduced plant vigour.
The cloning of the TDET1 gene and its use by means of gene transfer technologies offers considerable advantages with respect to conventional breeding techniques. It is possible to transfer genes suitable for agriculture between different species and to inactivate and/or engineer genes of the same species, even only in specific plant tissues. Furthermore gene transfer is a much more rapid technique than conventional breeding.
The cloning of the TDET1 gene now allows the production of engineered plants which exhibit all the favourable features of the hp mutation, with none of the undesirable side-effects. Such plants could belong to the Solanum genus, but those skilled in the art will recognize that, with no inventive effort, it is possible to transfer the gene, or parts thereof, subcloned in suitable expression vectors, into plants belonging to different genera, as e.g. but not limited to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species.
It has been well recognized that a diet rich in lycopene and other carotenoids, or their administration in the form of pills, produce favourable effects on human health. As a matter of fact, carotenoids have antioxidant properties, xcex2-carotene is a pro-vitamin A and lycopene is known to be an effective antitumoral agent (Bartley and Scolnik, 1995; Giles and Ireland, 1997; Hoffmann and Weisburger, 1997; Pappalardo et al., 1997; Pool-Zobel et al., 1997; Rock et al., 1997; Sharoni et al., 1997; Stahl and Sies, 1996). Therefore the engineered plants of the present invention can be advantageously utilized as a source of such compounds, either as fresh or processed foods or as nutraceuticals.
Furthermore hp-2 mutants have high levels of flavonoids such as anthocyanins (Von Wettstein-Knowles, 1968), which are also considered to be excellent antioxidants and which exhibit in some cases antitumoral properties (Fotsis et al., 1997; Rice-Evans et al., 1997). Furthermore some flavonoids exhibit a role in plant protection against pathogenic agents and UV light irradiation (Shirley,1996).
Therefore the manipulation of TDET1 activity can be used to modify carotenoid and/or flavonoid content in several plant species (e.g., tomato, pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, and pomaceae) for biotechnological uses in both the biomedical and agro-industrial sectors.
Furthermore the manipulation of TDET1 gene expression can be used to modify the anthocyanin and carotenoid content in ornamental species for the achievement of new colour variants (Griesbach, 1984).
In addition, because it has been recognized that a high content of carotenoids improves resistance to Norflurazon-type herbicides, a further application of the TDET1 gene is the production of transgenic plants by selection using herbicides rather than antibiotic compounds (Misawa et al., 1993).
Furthermore in the same plant it is possible to combine a modified TDET1 activity with mutations such as rin, nor and Nr, which interrupt the fruit ripening process, or with biosynthetic genes of the carotenoid biosynthesis pathway (Bartley and Scolnik, 1995), to obtain varieties exhibiting new qualitative characteristics for agro-industry.
The attainment of an hp mutant phenotype by means of a biotechnological approach can be carried out advantageously by means of the inhibition of TDET1 activity. Currently the best method for reducing gene expression is through the introduction, by gene transfer, of the antisense sequence of the same gene or part thereof under the control of appropriate regulatory sequences. The use of such techniques in plants has been carried out in several existing examples (Oeller et al., 1991; Penarrubia et al., 1992), but those skilled in the art will recognize that alternative techniques can be used, without departing from the scope of the present invention.
Furthermore, by using specific vectors such as, for example, but not limited to, pE8mGFP4, which contains regulatory sequences of the tomato E8 gene (FIG. 9) (Deikman and Fischer, 1988), the inactivation of the TDET1 gene can be specifically modulated in the tomato fruit. Those skilled in the art will recognize that other regulatory sequences, e.g., originating from the polygalacturonase gene (PG) (Nicholass et al., 1995), can be used in the place of the E8 gene promoter to obtain specific tissue modulation of TDET1 gene expression.
Within the scope of the present invention the term xe2x80x9clight hypersensitive phenotypexe2x80x9d means reduced plant growth associated with high levels of carotenoids and/or chlorophylls and/or flavonoids.
The term xe2x80x9cprotein or functional parts thereof, responsible for the light hypersensitive mutant phenotypexe2x80x9d means an amino acid sequence which, if structurally or otherwise altered, induces a light hypersensitive phenotype in a plant.
Therefore one object of the present invention is a nucleic acid comprising a nucleotide sequence encoding a protein, or functional parts thereof, which, if modified, is responsible for the light hypersensitive mutant phenotype in Solanum lycopersicum plants, said phenotype including reduced plant growth associated with high levels of carotenoids and/or chlorophylls and/or flavonoids. Nucleic acids encoding proteins which are homologous to proteins of the Arabidopsis COP/DET/FUS family, which when modified result in a light hypersensitive phenotype, are within the scope of the present invention.
Preferably the nucleic acid comprises the nucleotide sequence encoding the TDET1 protein, or functional parts thereof. More preferably the nucleic acid comprises a nucleotide sequence encoding the protein having the amino acid sequence of SEQ ID No. 2 or functional portions thereof. More preferably the nucleic acid has a nucleotide sequence comprised in SEQ ID No. 1, more preferably from nt. 149 to nt. 1720. Alternatively the nucleic acid has a nucleotide sequence complementary to SEQ ID No. 1, or parts thereof.
In one aspect of the invention the nucleic acid of SEQ ID No. 1 comprises a mutation which is able to induce the light hypersensitive phenotype; preferably at least a Cxe2x86x92T substitution in position 1640; alternatively the nucleic acid of SEQ ID No. 1 is deleted at least from nt. 1581 to nt. 1589.
A further object of the present invention is an expression vector including, under the control of an active and inducible plant promoter, the nucleic acid of the invention. Preferably the promoter is active only in some plant organs, more preferably in fruits. Preferred vectors are able to drive the transcription of an antisense RNA, for example pBIN-E8-HP2-AS1 and pBIN-E8-HP2-AS2.
A further object of the invention is the use of the vectors of the invention for producing transgenic plants which comprise the nucleic acid, under the control of specific regulating sequences, preferably in preselected plant organs. Plants can belong to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species.
A further object of the invention is a transgenic plant, or parts thereof, achievable by transformation with the vectors of the invention. Plants can belong to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species.
Plant genetic transformation techniques are known to those skilled in the art and comprise, but are not limited to transformation by Agrobacterium, electroporation, microinjection, or bombardment with DNA coated particles(Christou, 1996).
In a further aspect the invention includes a protein, or functional parts thereof, whose modification is responsible for the light hypersenstive phenotype in Solanum lycopersicum plants. Proteins homologous to the Arabidopsis COP/DET/FUS family are within the scope of the present invention, provided that, when modified, they result in a light hypersensitive phenotype. Preferably the protein comprises the amino acid sequence of SEQ ID No. 2 or parts thereof.
In one aspect of the invention the protein comprises a modification which is able to induce the light hypersensitive phenotype; preferably at least a modification in its C-terminal portion; more preferably a replacement of proline at position 498, most preferably serine as a substitute for proline; alternatively a deletion of at least one amino acid, preferably at least of three amino acids, more preferably from aa. 478 to aa. 480 of SEQ ID No. 2.